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Cardiovascular Research 2001 50(1):46-55; doi:10.1016/S0008-6363(00)00323-0
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Copyright © 2001, European Society of Cardiology

Altered expression of Bag-1 in Coxsackievirus B3 infected mouse heart

Tianqing Pengb, Teresa Saduskya, Yanwen Lia, Gary R. Coultona, Hongyi Zhanga,* and Leonard C. Archard

aMolecular Pathology Section, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, Exhibition Road, South Kensington, London, SW7 2AZ, UK
bKey Laboratory of Viral Heart Disease of Ministry of Public Health, Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Shanghai, PR China

* Corresponding author. Tel.: +44-207-594-3025; fax: +44-207-594-3022 h.zhang{at}ic.ac.uk

Received 19 September 2000; accepted 12 December 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objective: The mechanisms by which Coxsackie B viruses cause myocarditis or dilated cardiomyopathy are not well understood. This study examined changes in the expression of cardiac genes resulting from Coxsackievirus B3 (CVB3) infection of mice. Methods: Mice (five per group) were experimentally infected with CVB3 or mock-infected with diluent. Altered expression of genes was initially identified by cDNA array, and confirmed by semiquantitative RT-PCR, western blot and immunohistochemistry. Results: Forty-two up-regulated or down-regulated genes were observed in cDNA arrays carrying 588 known mouse genes. Among these, one down-regulated gene, Bag-1, known to be involved in inhibition of apoptosis and modulation of chaperone activity, was investigated further. Semiquantitative RT-PCR showed that Bag-1 expression was down-regulated by up to 30% in virus-infected mouse heart on day 7 compared to the mock-infected. Cell fractionation and western blot analysis confirmed that Bag-1 isoform p32 was predominant in the cytoplasm of mouse myocardium and down-regulated at 4 days or 7 days after CVB3 infection. In contrast, Bag-1 isoform p50 appeared to increase in the nuclear fraction of mouse heart at 7 days after infection. Down regulated expression and distribution of Bag-1 protein or evidence of apoptosis in the infected mouse heart was demonstrated by immunostaining or histochemistry (TUNEL assay), respectively. Conclusion: CVB3 infection induced differential expression of Bag-1 in cytoplasmic and nuclear fractions of mouse heart and apoptosis. This may be important in the pathogenesis of enterovirus heart muscle disease.

KEYWORDS Cardiomyopathy; Myocarditis


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Enteroviruses, particularly Coxsackie B viruses (CVB), are the most common cause of human viral myocarditis [1]. Viral infection can cause rapid and severe cardiac dysfunction. Patients may recover or experience progressive disease, leading ultimately to end-stage dilated cardiomyopathy (DCM), which requires transplantation. The concept that persistent enterovirus infection contributes to pathogenesis in a subset of human DCM cases was strengthened by detection of enteroviral genomic RNA in the heart of patients with DCM by molecular diagnostic approaches [2,3] and more recently by our in situ demonstration of enteroviral capsid protein VP1 in myocardial tissues from DCM patients [4]. CVB RNA can be detected in the hearts of as many as 30–50% of patients with DCM [5,6] and persistence of enteroviral RNA in heart is an independent predictor of poor prognosis [7] and involves an altered pattern of viral transcription in heart or skeletal muscle [8–10]. These findings indicate that enterovirus infection plays a role in the pathogenesis of DCM. However, the mechanisms by which enteroviruses cause myocarditis and progression to DCM are not well understood and there are no virus-specific preventive or therapeutic procedures available to protect humans against enterovirus induced heart muscle disease.

Although the immune response of the host undoubtedly plays an important role in the pathogenesis of viral heart disease [11], viral direct cytotoxicity was found to be crucial for organ pathology both during acute and persistent heart muscle infection. In cultured cardiomyocytes, infection with CVB3 induces a cytopathic effect and cell death [12] and the production of defective CVB3 RNA in the heart in a transgenic mouse model leads to the development of a heart disease resembling DCM in humans [13]. Enteroviral protease 2A directly cleaves dystrophin, and impairs its functions in the cytoskeleton [14]. In addition, apoptotic events may contribute to CVB induced pathogenesis. CVB3 infection induces caspase-3 activation in vitro [15] and apoptotic cells were detectable in inflammatory lesions as well as myocardial tissue outside inflamed areas, depending on the mouse strains and virus variants used [16–19].

Many host factors influence viral replication, viral persistence and disease chronicity [19,20]. The most important, however, is likely to be the host genetic background [21]. The src family kinase Lck (P56lck) has been reported recently to be essential host factor that influences replication and pathogenicity of CVB3 in vitro and in vivo but is not a general regulator of enterovirus pathogenicity [22], implying a complicated interaction of CVB with host cell in the development of viral heart muscle disease. It is postulated that susceptibility to CVB infection and the development of viral heart disease may be associated with expression of cellular genes involved in viral replication, viral persistence, signal transduction pathways, regulation of host gene transcription or translation and cardiac cell death or apoptosis [10,17,23,24]. Expression of these genes may be induced or altered by CVB infection and then play a role in the pathogenesis of CVB heart muscle disease. Identification of such cellular genes may allow the design of drugs that specifically interfere with or prevent the development of enterovirus heart muscle disease.

To identify molecular mechanisms of CVB heart muscle disease, we used duplicate cDNA arrays to detect changes in the pattern of cardiac gene expression in mouse myocardium, resulting from CVB infection. This allowed us to compare the expression of 588 known genes in CVB infected and mock infected mouse hearts.

In this article, we report altered expression of Bag-1 in mouse heart after CVB3 infection. Bag-1 is known to be involved in inhibition of apoptosis and modulation of chaperone activity [25,26]. Identification of differentially expressed cardiac genes may give new insights into molecular mechanisms of CVB heart muscle disease.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Animals and tissue processing
Five-week-old male SWR or MF-1 mice were obtained from Harlen (UK). They were randomly allocated with five in each group and housed in negative pressure isolators. SWR mice were used for analysis of gene profiles by cDNA array and further investigation of Bag-1 expression by RT-PCR, western blot and immunohistochemistry. MF1 mice were used to confirm the altered expression of Bag-1 at the mRNA and protein levels. The mice were inoculated with CVB3 in test groups or with diluent only (mock-infected) as controls, sacrificed at 4 or 7 days postinfection (p.i.) and the hearts were removed and processed as described previously [27]. The investigation conforms with the Animals (Scientific Procedures) Act 1986, UK.

2.2 Histopathology, in situ hybridization and immunohistochemistry
Formalin-fixed tissues were processed and embedded in paraffin following routine procedures. Two sections of 4-µm thick and two further sections deeper into paraffin tissue blocks (at least 40 µm apart from the first two) were cut from each tissue sample for histopathology, in situ hybridisation and immunohistochemistry. In situ hybridization for detection of CVB3 RNA using oligonucleotide probes (Table 1) labelled with DIG 3'-end oligonucleotide tailing kit was carried out according to manufacturer's instructions (Roche Diagnostics, UK). To detect CVB3 VP1 antigen with a monoclonal antibody using an Animal Research Kit (Dako, UK), or Bag-1 protein with the rabbit polyclonal anti-Bag-1 antibodies at dilution of 1:50 (C16, Santa Cruz Biotechnology, UK), immunostaining was performed according to our improved protocols [4,28].


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  		Table 1 Oligonucleotide probes for Coxsackieviral genomic RNA

 
2.3 Isolation of cellular mRNA or total RNA
All frozen myocardial samples from CVB3-infected or mock-infected mice were used for RNA isolation. The heart tissue was homogenised and mRNA isolation was carried out as described in the protocols for the Micro-FastTrackTM Kit (Invitrogen, USA) for cDNA array experiments. Total cellular RNAs were extracted with Tri Reagent (Sigma, UK) according to the manufacturer's instructions for RT-PCR. Contaminating chromosomal DNA was eliminated by treatment with RNase-free DNase I, followed by phenol–chloroform extraction.

2.4 Mouse cDNA array
The AtlasTM Mouse cDNA expression array I carrying 588 known mouse genes (Clontech Laboratories, USA) was used and RNA–cDNA hybridization to filters was carried out according to manufacturer's instructions. Briefly, mRNA was extracted from myocardial tissue pooled from five CVB3-infected or five mock-infected, age-matched SWR mice. Following cDNA synthesis and labelling with 32P dCTP, the same quantity of the mRNA extract was hybridised to duplicate filters. The hybridization signal was quantified by phosphorimaging and also imaged by autoradiography. Duplicate filters were compared and those genes showing significant differential expression were identified.

2.5 Semiquantitative reverse-transcriptase polymerase chain reaction
Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was performed as described [29,30] with some modifications: 5 µg of total RNA was used for reverse transcription in a 30-µl reaction mixture containing 1 µg of oligo (dT15) as primer, together with 100 U Superscript II reverse transcriptase II (Gibco, USA) according to the manufacturer's instruction. For PCR, 0.25–8 µl of the cDNA mixture was added to 50 µl of a master mix containing 200 µmol/l of each dNTP, 25 pmol of each specific primer, 1.5 mmol/l MgCl2, and 1 U of Taq DNA polymerase (Promega, USA). Negative controls including a water blank and no RT were included in every run. Each sample was amplified in three separate experiments in a thermal cycler (Perkin Elmer-Cetus). PCR products were separated on 1.5% agarose gel and transferred to nylon membranes with an alkaline buffer. Hybridization and detection were carried out using sequenced PCR products of Bag-1 gene or β-actin gene as probes, directly labelled with alkaline phosphatase for use in conjunction with chemiluminescent detection with CDP-Star (Amersham Pharmacia Biotech, UK) according to manufacturer's instructions. The exposed X-ray films were scanned and the density of the bands was computer-analysed by NIH 1.6 IMAGE software (National Institutes of Health). The relative intensity of bands for each mRNA was divided by the intensity of the β-actin internal control band. Semiquantitative RT-PCR detection was based on the optimal conditions for each set of primers, derived from calibration curves (Fig. 1) by which Bag-1 cDNA was exponentially amplified using 1 µl of the 30 µl reverse-transcription reaction products as template and 20 cycles. The same quantity of cDNA and 16 cycles were used for amplification of the β-actin sequence. The oligonucleotide primers for Bag-1 and β-actin gene are listed in Table 2.


Figure 1
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  		Fig. 1 Calibration curves for Bag-1 and β-actin RT-PCR. (A) cDNA dilution. (B) Number of cycles.

 

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  		Table 2 Primers for RT-PCR amplification of mouse Bag-1 and β-actin gene

 
2.6 DNA sequencing analysis
PCR products (Bag-1 and β-actin, respectively) were purified using a PCR product purification kit (Qiagen, Germany). Cycle sequencing was carried out on a ABI model 377 semiautomated DNA sequencer as described previously [31]. Sequence data was analysed using SEQUENCE ANALYSIS 2.1.2, SeqEd 1.0.3 (ABI) and AssemblyLignTM (Oxford Molecular Group, UK).

2.7 Subcellular fractionation and Western blotting
Frozen mouse myocardium was homogenised and lysed in solution A [0.15 mol/l NaCl, 1% (v/v) Nonidet P40, 0.5% sodium deoxycholate, 0.1% (w/v) SDS, 50 mmol/l Tris–HCl (pH 8.0)] containing protease inhibitors (complete tablet: 1697498, Roche Diagnostics UK). For protein localisation experiments, nuclear and non-nuclear fractions were prepared as described previously [32,33]. Briefly, heart tissue was homogenised gently in a glass tissue grinder with 20 volumes of 0.25 mol/l sucrose solution with protease inhibitors. Homogenates were centrifuged at 600 g for 10 min and the supernatants used as cytosolic fractions. The pellets containing nuclei were washed twice in the same buffer and finally resuspended in buffer A by shaking vigorously for 10 min. Protein concentrations were measured by the Bradford assay kit and compared with known concentration of bovine serum albumin (Bio-Rad, USA).

For Western blotting, aliquots containing 30 µg of protein were subjected to SDS–PAGE using 12% gels, followed by electrotransfer to ECL membranes. Immunodetection was accomplished using 1:400 (v/v) of anti-Bag-1 antibody C16, followed by incubation with horseradish peroxidase-conjugated secondary antibody (Dako). Detection was performed using an ECL chemiluminescence detection method (Amersham Pharmacia Biotech). Bag-1 levels were measured by scanning microdensitometry on X-ray films and computer-analysed using the NIH 1.6 IMAGE software, normalised to the amount of loaded proteins.

2.8 TUNEL staining
Apoptotic cells in the mouse myocardium were detected by using the TUNEL method. An in situ apoptosis detection kit, Apop®Tag Plus Peroxidase, was supplied by Intergen (UK) and the detection experiment was carried out as described in the manufacturer's manual. The number of apoptotic nuclei (detected as brown color) per cross-sectional slice of the mid-ventricular wall was counted under a microscope and described as means±S.D.

2.9 Statistical analysis
The Mann–Whitney nonparametric test was used (INSTAT 2.01 software) to analyse the statistical significance of differences between CVB3-infected and mock-infected mice. A P value of <0.05 between groups was regarded as statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 Evidence of myocarditis and coxsackievirus B3 infection in mouse heart
Histological examination of H&E stained sections showed the presence of multiple foci of tissue necrosis with inflammatory infiltration in MF-1 mouse heart at 4 days p.i. and more severe pathological changes at 7 days p.i., similar to those seen in the characterised SWR mouse heart (Fig. 2). To confirm and localise CVB3 infection in heart tissue, in situ hybridization for viral RNA and immunohistochemistry for viral capsid protein VP1 were performed on paraffin sections. Positive signals for viral RNA or VP1 were observed in the myocardium of infected MF-1 and SWR mice (Fig. 2). Most VP1 signals were in or around cytopathic lesions, suggesting direct virus-mediated pathogenesis. Pancreatic tissue sections from infected mice were used as positive controls for detection of viral RNA and VP1 protein (Fig. 2). No positive signals for viral RNA or VP1 were observed in tissue from age-matched, mock-infected mice.


Figure 2
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  		Fig. 2 Photomicrographs of mouse heart and pancreas. Tissue sections from CVB3 or mock-infected MF1 or SWR mice 7 days p.i. were processed for histology (H&E staining) or detection of enterovirus capsid protein VP1 (counter-stained with hematoxylin) or viral genomic RNA (see Methods). (A) Myocardial section from a mock-infected MF-1 mouse, showing normal histology. Myocardial sections from a CVB3-infected MF-1 (B) or SWR (C) mouse, showing myocarditis. Detection of VP1 in the pancreatic (D) or myocardial (E) section from CVB3-infected MF1 or SWR mouse (F) by immunohistochemistry (yellow-brown signal). Detection of CVB3 RNA in the pancreatic or myocardial section from CVB3-infected MF1 mouse (G,H) or SWR mouse (I) by in situ hybridization (dark purple signal). (Magnification: 200x).

 
3.2 cDNA expression profiles of mouse cardiac genes
The results of mouse cDNA array experiment showed that the expression of the housekeeping gene, β-actin, was similar in CVB3 infected mice and age-matched, mock infected mice, suggesting that expression of β-actin is an appropriate standard for identifying differential expression of other genes. Thus, following normalization to β-actin hybridisation signals, the patterns of gene expression in CVB3 infected mice were compared with those of mock-infected mice. Forty-two genes up-regulated or down-regulated at the mRNA level were identified (data not shown). One gene, Bag-1, was down-regulated and was chosen for further investigation.

3.3 Quantification of differentially expressed Bag-1 mRNA
Semiquantitative RT-PCR was performed to confirm and quantify the differential expression of Bag-1 gene observed in hybridization to cDNA arrays. Amplified Bag-1 and β-actin gene products were confirmed by DNA sequencing (data not shown) and in turn used as hybridization probes for Southern blots to identify Bag-1 or β-actin RT-PCR products from CVB3-infected or mock-infected mouse heart. The relative Bag-1 mRNA levels were determined by the intensity of Bag-1 bands relative to the intensity of the housekeeping gene β-actin bands in particular samples. CVB3 infection of MF-1 mice for 7 days resulted in a pronounced decrease in Bag-1 mRNA level in the heart, about to 30% of that in mock-infected heart (Fig. 3), similar to that of SWR mice (data not shown). Water blank and no RT controls were negative.


Figure 3
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  		Fig. 3 Quantification of Bag-1 transcription in MF-1 mouse heart. Bag-1 mRNA from CVB3 or mock-infected mice heart 7 days p.i. were analyzed by semiquantitative RT-PCR using β-actin as internal control (see Methods). Southern hybridization of Bag-1 and β-actin RT-PCR products (upper figure) and the relative Bag-1 mRNA levels expressed in Bag-1/β-actin ratio (lower figure). Nos. 1–5: mock-infected mice. Nos. 6–9: CVB3 infected mice.

 
3.4 Down-regulated expression of Bag-1 protein in heart from CVB3 infected mouse
It has been reported that two Bag-1 isoforms, p50 (50 kDa) and p32 (32 kDa), arise in mouse by use of alternative translation initiation sites within a common Bag-1 mRNA [34]. The polyclonal anti-Bag-1 antibody C16 used in this study recognises both mouse Bag-1 p50 and p32. In Western blots of whole tissue lysates from normal MF-1 mouse heart, a strong p32 signal and a weak p50 signal were seen; hearts from CVB3 infected mice showed a significant decrease in p32 protein level at both 4 and 7 days p.i., being reduced to 50–60% of that of mock infected mice (Fig. 4), similar to that observed in the SWR mice (data not shown).


Figure 4
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  		Fig. 4 Quantification of expression of Bag-1 protein in MF-1 mouse heart. Tissue lysates from mock- or CVB3-infected mouse heart were analysed by Western blot using anti-Bag-1 antibody C16. Total Bag-1 proteins from myocardial tissues are shown in the upper figure and the relative Bag-1 p32 protein levels in the lower figure. cvb/4d, infection with CVB3 for 4 days; cvb/7d, infection with CVB3 for 7 days; con: mock infected. (*, P<0.01, n = 5).

 
Bag-1 protein levels and cellular distribution in myocardium were also assessed by immunohistochemistry. The results show cytoplasmic and nuclear staining of Bag-1 in cardiomyocytes and infiltrating lymphocytes of both MF-1 and SWR mice (Fig. 5). Bag-1 immunostaining signals in CVB3-infected mouse myocardium are significantly weaker than in mock-infected mice, further confirming down-regulation of Bag-1 after CVB3 infection.


Figure 5
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  		Fig. 5 Photomicrographs of mouse heart. Tissue sections from CVB3 or mock-infected MF1 or SWR mice 7 days p.i. were processed for detection of Bag-1 protein levels, distribution in myocardium and detection of apoptotic cells by TUNEL staining (see Methods). The myocardial section from mock infected MF-1 mouse (A) or SWR mouse (C) 7 days p.i. The myocardial section from CVB3-infected MF-1 mouse (B) or SWR mouse (D) 7 days p.i., showing nuclear staining of Bag-1 (yellow-brown signal) in myocytes (arrows in C) and infiltrating lymphocytes (arrows in D), and reduced cytoplasmic staining of Bag-1 protein. Apoptotic infiltrating lymphocytes (E) and myocytes (F) in a myocardial section from a CVB3-infected MF-1 mouse are detected by TUNEL staining (yellow-brown signal, arrows showing nuclear localization). (Magnification: 400x for A and B, 200x for C–F).

 
3.5 Isoform-specific expression of Bag-1 proteins in subcellular fractions of mouse heart
Nuclear and cytosolic cell fractions from MF-1 mouse heart were examined separately, to investigate intracellular changes in Bag-1 protein resulting from CVB3 infection. Western blot analysis showed that Bag-1 p32 was predominantly found in the cytoplasm and p50 in the nucleus of both CVB3-infected and mock-infected mouse heart (Fig. 6). The expression of p32 was decreased in cytosolic fraction of mouse heart tissue at both 4 and 7 days p.i., as observed in whole cell lysate. In contrast, Bag-1 p50 increased in the nuclear fraction of mouse heart tissue 7 days p.i. No significant difference of Bag-1 p50 expression in nuclear fractions of mouse heart tissues was observed between mouse heart infected with CVB3 or mock infected heart at 4 days p.i.


Figure 6
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  		Fig. 6 Isoform-specific expression of Bag-1 proteins in subcellular fractions of mouse heart. Detection of Bag-1 isoforms in cytosolic and nuclear fraction of mock-infected or CVB3-infected MF-1 mice by western blot using anti-Bag-1 antibody C16 are represented in (A, 4 days p.i.) or (C, 7 days p.i.). The relative Bag-1 p32 or p50 protein levels were calculated from five individual animals in each group and shown in (D, 4 days p.i.) or (E, 7 days p.i.). Bag-1 subcellular distribution is shown in (B) p32 being predominant in the cytoplasm and p50 in the nucleus of myocardium. cvb/4d, infection with CVB3 for 4 days; cvb/7d, infection with CVB3 for 7 days; con, mock infected. nu-F: nuclear fractions; cyto-F, cytosolic fractions. (*, P<0.01).

 
3.6 Presence of apoptotic cells in myocardial tissues of CVB3 infected mice
The TUNEL assay was employed for detection of apoptotic cells in heart tissue of both MF-1 and SWR mice. Positive signals were observed in some of cardiomyocytes or infiltrating lymphocytes in myocardial tissues of CVB3 infected mice (Fig. 5E and F). In CVB3-infected MF1 mice, the number of TUNEL-positive nuclei in a mid-ventricle cross-section was 15.38±2.02 (n = 5) at 4 days p.i. and 12.20±1.75 (n = 5) at 7 days p.i. In CVB3-infected SWR mice, it was 15.40±2.70 (n = 5) at 7 days p.i. In comparison, there were significantly less apoptotic cells in mock-infected MF1 (4.00±0.41, P<0.05, n = 5) or SWR (5.25±1.71, P<0.05, n = 5) mice on day 7. These results of measuring apoptosis event in the mouse heart following CVB3 infection correlate with down-regulation of Bag-1 expression.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
Bag-1 was originally cloned from a mouse library as a Bcl-2 binding protein and shown to have anti-apoptotic activity [26]. It also forms complexes with several other proteins to modulate their activities [35–38]. The mechanism by which Bag-1 influences the function of such diverse proteins may be attributed to its ability to bind heat shock protein directly, which otherwise interacts with multiple protein targets in cells [25]. At least two Bag-1 isoforms, p50 and p32, have been reported in mouse cells as a result of alternative initiation of translation within a common mRNA [34], and have distinct subcellular localization which may allow Bag-1 isoforms to fulfil different functions. In this report, decreased Bag-1 mRNA and Bag-1 protein levels in CVB3 infected hearts were demonstrated in two different mouse strains, one inbred (SWR H-2q) and the other outbred (MF-1). In subcellular fractions, Bag-1 p32 isoform decreased in the cytosolic fraction of myocardium of CVB3 infected mouse, whereas Bag-1 p50 isoform increased in the nuclear fraction of mouse myocardium at 7 days p.i., but not at 4 days. This change may result from lymphocyte infiltration in the mouse myocardium following CVB3 infection as (1) there are more infiltrating lymphocytes in mouse myocardium at 7 days p.i. compared to 4 days p.i. and (2) a majority of infiltrating lymphocytes show nuclear immunostaining of Bag-1 protein in CVB3 infected mouse heart.

Apoptosis may be an important mechanism in the development of enterovirus heart muscle disease. For example, CVB3 infection elevates the concentration of myocardial cytosolic free calcium and induces apoptosis in cultured rat cardiomyocytes early in infection [39]. It has been reported that the CVB3 non-structural protein 2B can modify plasma membrane and endoplasmic reticulum permeability, thus inducing an increased levels of cytosolic free calcium [40]. Elevation of intracellular free Ca2+ levels can activate calcineurin that in turn dephosphorylates BAD, a proapoptotic member of the Bcl-2 family, thus enhancing BAD heterodimerization with Bcl-xL and promoting apoptosis [41]. More recently, CVB-induced apoptosis of cardiomyocytes was reported by Henke et al. [17] who found that the transcription of murine proapoptotic protein Siva, involved in CD27/CD70-mediated apoptosis, is strongly induced in myocardium of CVB3-infected mice. Moreover, CVB3 capsid protein VP2 interacts with the Siva protein, suggesting a molecular mechanism through which apoptotic events may contribute to the pathogenesis of CVB3-induced heart muscle disease.

It has been reported that Bag-1 interacts with multiple cellular targets and suppresses apoptosis in several systems. For example, Bag-1 p32 binds to Bcl-2 and enhances Bcl-2 suppression of Fas-mediated, staurosporine-induced and cytotoxic T-lymphocyte-induced apoptosis [34]. Binding of p32 Bag-1 to Raf-1 and subsequent activation of Raf-1 cooperates with Bcl-2 in suppressing apoptosis [36]. Bag-1 p32 enhances protection from apoptosis induced by hepatocyte growth factor receptor and platelet-derived growth factor receptor [35], and inhibits retinoic-acid-induced apoptosis [37]. Bag-1 p32 also binds to SIAH-1, which can induce apoptosis and promote expression of the tumour suppressor p53–p21waf1 inducible gene, and this binding is responsible for inhibition of the growth arrest effect of p53 [42]. Down-regulation of Bag-1 by CVB3 infection can therefore promote apoptosis as shown in the current study and such a reduction of anti-apoptotic proteins may be an important mechanism by which these viruses cause cardiomyocyte death. In addition, decreased mRNA levels of another Bcl-2 interacting anti-apoptotic protein, Nip21, were observed in CVB3 infected mouse heart by a different laboratory [43].

Bag-1 is known to interact with and modulate the activities of chaperones such as HSP70/HSC70 [25]. Previous studies have indicated that HSP70 blocks iNOS expression by binding its transcription factor, NFkB, thus preventing its translocation to nucleus [44], and also controls TNF-{alpha} production and activity [45]. Both iNOS [46] and TNF{alpha} [47,48] have been shown to play a role in the pathogenesis of enterovirus heart disease. Induction of iNOS results in the production of large amounts of NO, which inhibits myocyte contractility, is directly cytotoxic and has a negative inotropic action [49,50]. TNF{alpha} depresses myocardial contractile function by disruption of calcium handling, direct cytotoxicity, oxidant stress, disruption of excitation–contraction coupling and cardiac myocyte apoptosis [45]. It is possible that alterations of Bag-1 expression indirectly modulate the production and activity of TNF{alpha} and iNOS via its interaction with chaperones.

Bag-1 has also been found to participate in regulation of the nuclear receptor family which acts as signal proteins. Functional differences between Bag-1 isoforms in the regulation of signal transduction pathways have been observed, possibly due to differential effects on Hsc70 chaperone function [51]. Altered expression of Bag-1 isoforms could affect the DNA binding and transactivating activities of the glucocorticoid receptor [38], the androgen receptor [52] and the retinoic acid receptor [37]. Net effects of Bag-1 isoforms on DNA binding and transactivating activities via signalling proteins would influence a variety of other cellular processes, pertinent to viral heart disease.

It is intriguing to find altered expression of Bag-1 in the heart of CVB3 infected mouse. Further investigation of pathways downstream of Bag-1 will improve our understanding of how CVB infection exerts its effects on cell survival, proliferation and differentiation and are likely to provide a novel insight into the molecular mechanisms of viral heart disease.

Time for primary review 24 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
This work was supported by the British Heart Foundation and National Natural Scientific Foundation of China (39970309).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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